Journal: Scientific Reports
Article Title: Human Sialidase Neu3 is S-Acylated and Behaves Like an Integral Membrane Protein
Figure Lengend Snippet: Neu3 behaves as an integral membrane protein. ( a ) Soluble (S, first lane) and membrane (P, second lane) fractions of NEU3 -transfected cells were obtained as described in Fig. 1a . The latter fraction was then incubated for 40 min on ice with Tris buffer (control) or Na 2 CO 3 at pH 11.5 in order to extract peripheral membrane proteins. After treatment, membranes were collected by ultracentrifugation, and membrane-bound (P, fourth and sixth lanes) and solubilized (S, third and fifth lanes) proteins were analyzed by Western blotting using anti-Neu3 antibody. K-Ras and Cav-1 were used as markers of an extractable and a non-extractable membrane protein, respectively. Tub was used as a marker of a soluble protein. ( b ) Intact NEU3 -transfected cells were treated with (+) or without (−) 100 µg/ml proteinase K (PK) for 30 min at 37 °C. The remaining proteins were run on a SDS-PAGE gel and Western blotted with anti-c-Myc and anti-Neu3 antibodies. Transferrin Receptor (TfR) was used as a marker of a PK-accessible protein, whereas Cav-1 and sialyltransferase ST3Gal-II were used as markers of proteins not accessible to PK. The molecular masses in kDa of the standard proteins are shown on the left. ( c ) Antibody accessibility in permeabilized and non-permeabilized cells. The signal of the indicated antibodies were analyzed after fixation in cells treated with 0.1% saponin in PBS/BSA (permeabilized) or with PBS/BSA alone (non-permeabilized). Cav-1 was used as a marker of an inner membrane protein detected only after cell permeabilization, and GPI-YFP was used as a marker of an outer membrane protein accessible by antibody without permeabilization. Cells nuclei were stained blue with Hoechst dye. ( d ) Cell surface proteins exposed to the extracellular environment of NEU3 -transfected cells were biotinylated and isolated by streptavidin agarose pull-down. Biotinylated (B) and non-biotinylated (NB) fractions were analyzed by Western blotting. Endogenous marker Cav-1 was used as control of a membrane protein not exposed to the extracellular environment. Densitometric analysis indicating the percentage of Neu3 in each fraction is shown. ( e ) Kyte-Doolittle hydrophobicity plot for Neu3 sequence using a window size of 19. The red line corresponds to a hydrophobicity score of 1.6 and designates an empirical cutoff value for α-helix transmembrane segments of ~19 amino acids long.
Article Snippet: Subcellular fractionation Cells were washed with cold PBS and harvested by scraping in 5 mM Tris/HCl pH 7.0 (buffer T) supplemented with protease inhibitor cocktail (PIC) (Sigma-Aldrich, St Louis, MO, USA).
Techniques: Transfection, Incubation, Western Blot, Marker, SDS Page, Staining, Isolation, Sequencing